Microbiological growth media and methods of using the same

ABSTRACT

The present invention features general-purpose microbiological growth media capable of supporting growth of microorganisms on membranes. The media contain casein digest, soybean digest, animal tissue digest, yeast extract, dextrose, a phosphate buffer, hemin, and L-cystine. The invention features an all-purpose microbiological growth media that can support the growth of anaerobes, molds, injured spores, and general aerobic bacteria to a greater extent than other media.

FIELD OF THE INVENTION

In general, the present invention relates to microbiological growthmedia and methods of their use,

BACKGROUND

In many industries, particularly the food, beverage, healthcare,electronic, and pharmaceutical industries. it is essential to analyzesamples for the degree of contamination by microorganisms, such asbacteria, yeasts, or molds rapidly. In particular, pharmaceutical andbiologics companies are required to test sterile: products for thepresence of microbiological contaminants. The traditional test, asdescribed in the United States Pharmacopeia monograph <71>, is agrowth-based assay that uses trypticase soy broth (TSB) at 22.5° C. andfluid thioglycollate medium (FTM) at 32.5° C. TSB is a general purposegrowth medium. used to detect yeasts, molds, and aerobic bacteria, FTMhas aerobic and anaerobic layers and is used to detect obligateanaerobes as well as aerobic bacteria. The intent for this combinationof growth conditions is to grow as many organisms as possible: While FTMhas the ability to grow anaerobes, it is a poor general purpose mediumwith limited growth promotion properties for many species. In addition,FTM has a limited ability to support the growth of microorganisms on asurface, particularly, of anaerobic organisms.

Other known growth media, such as Schaedler media, are specialized tosupport growth of certain microorganisms. For example, Schaedler mediaare optimized to support growth of human pathogens, whereas testingsterile products requires general media capable of supporting growth ofas many microorganisms as possible.

There remains a need for a general growth medium that is capable ofsupporting growth of aerobic and anaerobic bacteria, in particular, onmembranes.

SUMMARY OF THE INVENTION

The invention features an all purpose microbiological growth media thatcan support the growth of anaerobes, molds, injured spores, and generalaerobic bacteria to a greater extent than other media. The inventionfeatures a composition containing casein digest, soybean digest, animaltissue digest, yeast extract, dextrose, a phosphate buffer (e.g.potassium phosphate buffer), hemin, and L-cystine.

In a first aspect, the composition is a solid, e.g., a powder, at 22° C.Embodiments of compositions of the first aspect are summarized inTable 1. The amounts are provided in terms of grams of an individualcomponent relative to a kilogram of the total composition, and oneskilled in the art will understand that the total amounts of each of theindividual components will not exceed 1 kg.

TABLE 1 Non-limiting 1^(st) Range 2^(nd) Range 3^(rd) Range ExampleComponent (g/kg) (g/kg) (g/kg) (g/kg) Casein digest 1-500 50-400 100-300245.3 Soybean digest 0.5-300  10-200  10-100 43.8 Animal tissue digest1-500 50-400 100-300 219 Yeast extract 1-500 50-400 100-300 219 Dextrose1.500 50-400 100-300 255 Hemin 0.1-2    0.2-1   0.3-0.5 0.4 L-cystine4-80  8-40 12-20 17.5 SUM: 1000 1000 1000 1000

In particular embodiments of the first aspect, the compositions recitedin Table 1 further contain a phosphate buffer, e.g., a mixture ofdipotassium hydrogen phosphate or a hydrate thereof and potassiumdihydrogen phosphate or a hydrate thereof. In yet other embodiments, theamount of the phosphate buffer, e.g., potassium phosphate buffer,present in the composition of the first aspect is sufficient to providea buffer capacity of from 0.1 mmol/(pH unit) to 100 mmol/(pH unit),e.g., 1 mmol/(pH unit) to 50 mmol/(pH unit), 2 mmol/(pH unit) to 20mmol/(pH unit), or 3 mmol/(pH unit) to 10 mmol/(pH unit), in a mediumgenerated by dissolution or suspension of the composition of Table 1 andthe phosphate buffer in an aqueous medium (e.g., purified water, sheepblood (e.g., defibrinated sheep blood or laked sheep blood), or both).In other embodiments, phosphate buffer, e.g., potassium phosphatebuffer, is present in an amount sufficient to produce a pH of 7.3±0.5 ina liquid or gel composition (e.g. , PH of 7.3±0.2).

In some embodiments of the first aspect, the composition furthercontains a gelling agent. When the composition contains the gellingagent, the composition contains between about 10 g/kg and about 800g/kg, preferably between about 100 g/kg and about 600 g/kg, and morepreferably between about 250 g/kg and about 450 g/kg of the gellingagent (e.g., agar) (e.g., about 350 g/kg of a gelling agent). In someembodiments, the gelling agent is agar. In other embodiments, thegelling agent is gellan, sodium alginate, xanthan gum, guar gum,polyacrylamide, or Eladium™.

In certain embodiments of the first aspect, the composition furthercontains a surfactant. The composition of the first aspect can containbetween about 0 g/kg and about 190 g/kg of the surfactant, e.g., betweenabout 1 g/kg and about 60 g/kg, between about 4 g/kg and about 40 g/kg,or about 20 g/kg of the composition shown in Table 1. The surfactant ispreferably a polysorbate (e.g., polysorbate 20, also known as Tween®20).

In a second aspect, the composition is a liquid at 22° C. and, in athird aspect, the composition is a gel at 22° C. The liquid or gel willinclude sufficient water or other aqueous solution or suspension to formthe liquid or gel. In some embodiments, the liquid or gel compositionhas a PH of 73±0.5 (e.g., PH of 7.3±0.2).

Examples of compositions of the second or a third aspect are provided inTable 2. The amounts are provided in terms of grams of an individualcomponent relative to a kilogram of the total composition (includingsolvent and gelling agent, if present), and one skilled in the art willunderstand that the total amounts of all components will not exceed 1kg.

TABLE 2 Non-limiting 1^(st) Range 2^(nd) Range 3^(rd) Range ExampleComponent (g/kg) (g/kg) (g/kg) (g/kg) Casein digest 0.1-50 1-20 2-10 5.6Soybean digest 0.05-30  0.1-10  0.2-3   1 Animal tissue 0.1-50 1-20 2-105 digest Yeast extract 0.1-50 1-20 2-10 5 Dextrose 0.1-50 1-20 2.10 5.8Surfactant  0-19 0.05-5    0.1-1   0.5 Hemin  0.005-0.015 0.007-0.012 0.009-0.011  0.01 L-cystine 0.01-0.5  0.01-05   0.01-0.5  0.4

In certain embodiments, the composition of the second or third aspectcontains from 5 g to 100 g of the composition of Table 1 per kilogram ofaqueous medium (e.g., purified water, sheep blood (e.g., defibrinatedsheep blood of laked sheep blood), or both), e.g., from 10 g to 50 g,from 20 g to 30 g, or about 23 g of the composition in Table 1.

In other embodiments of the second or third aspect, the compositionsrecited in Table 2 further contain a phosphate buffer, e.g., potassiumphosphate buffer. In yet other embodiments, the amount of the phosphatebuffer, e.g. potassium phosphate buffer, present in the composition ofthe second or third aspect is sufficient to provide a buffer capacity offrom 0.1 mmol/(pH unit) to 100 mmol/(pH unit), e.g., 1 mmol/(pH unit) to50 mmol/(pH unit), 2 mmol/(pH unit) to 20 mmol/(pH unit), or 3 mmol/(pHunit) to 10 mmol/(pH unit).

In particular embodiments of the second or third aspect, the compositionfurther contains sheep blood (e.g., defibrinated sheep blood or lakedsheep blood). The concentration of sheep blood in the composition may beless than about 200 mL/kg, and preferably less than about 100 mL/kg(e.g., about 50 mL/kg). In particular embodiments, red blood cells inthe sheep blood are lysed (e.g., laked sheep blood or defibrinated sheepblood treated to lyse red blood cells).

In particular embodiments of the second or third aspect, the compositionfurther contains a surfactant. The composition of the second or thirdaspect can contain between 0 g/kg and 19 g/kg of the surfactant. e.g.,between about 0.05 g/kg and about 6 g/kg, between about 0.1 g/kg andabout 4 g/kg, or about 0.5 g/kg of the composition shown in Table 2. Thesurfactant is preferably a polysorbate (e.g., polysorbate 20, also knownas Tween® 20).

In certain embodiments of the second aspect and in the third aspect, thecomposition contains a gelling agent. It will be understood that aliquid may include a gelling agent in an amount insufficient to gel.Examples of gelling agents include agar, gellan, sodium alginate,xanthan gum, guar gum, polyacrylamide, and Eladium™. The concentrationof agar or Eladium™ in the composition is, for example, between about 5g/kg and about 25 g/kg (e.g., about 13.5 g/kg); the concentration ofgellan in the composition is, for example, between about 1.0 g/kg andabout 13 g/kg (e.g., about 6.8 g/kg: the concentration of xanthan gum orsodium alginate in the composition is, for example, between about 3.4g/kg and about 17 g/kg (e.g., about 9 g/kg): the concentration ofpolyacrylamide in said composition is, for example, between about 50g/kg and about 200 g/kg (e.g. about 150 g/kg); and the concentration orguar gum in said composition is, for example, between about 10 g/kg andabout 40 g/kg (e.g., about 21 g/kg).

In certain embodiments of the first through third aspects, the culturemedium may further include an antibiotic, e.g., for susceptibility orresistance testing or for selection of resistant cells. In otherembodiments of the first through third aspects, the growth mediumfurther includes a disinfectant neutralizer. Examples of disinfectantsto be neutralized include alcohols, hypochlorite, hydrogen peroxide,acetic acid, peroxyacetic acid, quaternary ammonium compounds phenolics,iodine, chlorine preparations, mercurials, formaldehyde, andglutaraldehyde. Examples of neutralizers include histidine, thiosulfate,polysorbate 80, and/or lecithin. Other neutralizers Includes bisulfite,glycine, divalent cations (e.g., Mg²⁺ or Ca²⁺) and thioglycollate.

In a fourth aspect, the invention features a method of culturing apopulation of cells by contacting the population of cells with thecomposition according to the second or third aspect of the inventionunder conditions supportive of growth of the population of cells.

In some embodiments of the fourth aspect, the population of cells isdisposed on one side of a permeable membrane with the other side of thepermeable membrane being in contact with the composition according tothe second or third aspect of the invention. Permeable membranes will beporous or otherwise capable of allowing transport of growth medium fromone side to the other.

In certain embodiments of the fourth aspect, the population of cellsincludes aerobes. In other embodiments, the population of cells includesanaerobes (e.g., an obligate anaerobe). In particular embodiments of thefourth aspect, the population of cells include cells belonging to agenus selected from the group consisting of Acinetobacter (e.g.,Acinetobacter lwofii), Aspergillus(e.g., Aspergillus brasillerisis orAspergillus fumigates), Bacillus(e.g., Bacillus clausii, Bacillusidriensis, Bacillus licheniformis, or Bacillus substilis),Corynebachterium (e.g., Coryneacterium tuberculostearicum orCorynebacterium xerosis), Dermacoccus(e.g., Dermacoccusnishiomlyaensis). Escherichia (e.g. Escherichia coli), Exserchilum(e.g., Exserohilum rostratum), Kocuria rhizophila), Methylobacterium(e.g., Methylobacterium radiotoferans), Micrococcus (e.g., Micrococcusluteus), Paenibacillus (e.g., Paenibacillus glucanolyticus), Penicillium(e.g., Penicillium chrysogenum or Penicillium notatum),Propionibacterium (e.g., Propionibacterium acnes), Pseudomonas (e.g.,Pseudomonas aeruginosa), Staphylococcus (e.g., Staphylococcus aureus,Staphylococcus epidermidis, or Staphylococcus hominis), Streptococcus(e.g., Streptococcus pyogenes), and Streptomyces (e.g., Streptomyceshalstedit). In some embodiments, the Bacillus is oxidatively stressed.

In some embodiments of the fourth aspect, the population of cells is ina sample. The sample may contain fluids or tissues obtained from amulticellular organism (e.g., a bodily fluid or tissue of an animal(e.g., a human or a non-human vertebrate)). The sample may be obtainedfrom the respiratory, urogenital, digestive, or reproductive tract,central nervous system, urine, skin, mucus, blood, plasma, serum, lymph,cerebrospinal fluid, saliva, wound tissue, wound exudate, biopsy, feces,or a solid tissue, or a derivative thereof. In certain embodiments, thesample is a blood or urine sample. The sample can also be derived from aplant or fungus. The sample may be obtained by sampling environmentalair, soil, or water, surfaces, objects, or organisms exposed to theenvironment. The sample may be obtained from raw, finished, orin-process material in the manufacture of pharmaceutical, cosmetic,blood, or other products for topical or internal use in humans oranimals; raw, in-process, or finished material in the manufacture offoods, beverages, or nutritional supplements (e.g., vitamins or herbalextracts); raw, in process, or finished material in the manufacture ofmedical or in vitro diagnostic devices; chemical products: industrialsurfaces; instrumentation; and machinery. The sample may be treated toliquefy and/or homogenize it prior to the contacting step. Additionallyor alternatively, prior to the contacting step, the sample may betreated to remove substances or objects other than the population ofcells, e.g., by filtration or sedimentation.

In a specific embodiment, the method is a sterility test that contactsthe sample with three separate aliquots of the culture medium, oneincubated aerobically at room temperature, e.g., about 22° C., oneincubated aerobically at an elevated temperature, e.g., about 32.5° C.,and one incubate anaerobically at an elevated temperature, e.g., about32.5° C.

In a fifth aspect, the invention features a method of preparing thecomposition according to the second aspect of the invention. The methodinvolves:

i) autoclaving a mixture containing purified water, casein digest,soybean digest, a phosphate buffer, dextrose, animal tissue digest,yeast extract, hemin, and L-cystine;

ii) optionally cooling the mixture to room temperature;

iii) optionally adjusting pH to 7.3±0.2 by adding sterile potassiumhydroxide or hydrogen chloride to the mixture; and

iv) adding sheep blood to the mixture.

In some embodiments of the fifth aspect, after step iv), the methodfurther includes step v) holding the temperature of the mixture at about65° C. until the color of the mixture changes from red to brown (e.g.,when sheep blood is defibrinated sheep blood). When laked sheep blood isused, step iv) may occur at or below 45° C.

In particular embodiments of the fifth aspect, the quantities of theingredients of casein digest, soybean digest, a phosphate buffer,dextrose, animal tissue digest, yeast extract, hemin, and L-cystine arethose described in the first aspect of the invention. In certainembodiments of the fifth aspect, the quantity of sheep blood is the sameas that described in the second or third aspect of the invention.

In certain embodiments of the fifth aspect, the mixture in step i)further contains a gelling agent, e.g., agar, gellan, sodium alginate,xanthan gum, guar gum, polyacrylamide, or Eladium™.

In some embodiments of the fifth aspect, the mixture in step i) furthercontains a surfactant, e.g., a polysorbate (e.g. polysorbate 20).Alternatively, the surfactant can be addedd after step i) (e.g., afterstep iv) or v)).

In other embodiments of the fifth aspect, the final composition istransferred into a storage vessel (e.g., a bottle, a jar, a vial, anampoule, or a cassette (e.g., a cassette, such as the cassette describedin WO 2013/070730)). The filled sterile storage vessel can beγ-irradiated to sterilize the medium. The dosage of sterilizing γradiation can be greater than 10 kGy, e.g., between 10 kGy and 50 kGy,between 10 kGy and 40 kGy, between 10 kGy and 30 kGy, or between 10 kGyand 20 kGy (e.g., between 12 kGy and 19 kGy).

In some embodiments of any aspect of the invention, the composition doesnot contain tris(hydroxymethyl)aminomethane. In particular embodimentsof any aspect of the invention, the composition does not include addedsodium. For example, none of the dextrose, phosphate buffer, hemin, andL-cystine includes sodium.

In certain other embodiments of any aspect of the invention, thephosphate buffer includes one or more of tripotassium phosphate or ahydrate thereof, dipotassium hydrogen phosphate or a hydrate thereof,and potassium dihydrogen phosphate or a hydrate thereof. In particularembodiments, the phosphate buffer includes one or more of dipotassiumhydrogen phosphate or a hydrate thereof and potassium dihydrogenphosphate or a hydrate thereof. In other embodiments, the phosphatebuffer is a mixture of dipotassium hydrogen phosphate or a hydratethereof and potassium dihydrogen phosphate or a hydrate thereof.

In some embodiments of the first aspect of the invention, thecomposition consists of casein digest, soybean digest, animal tissuedigest, yeast extract, dextrose, a phosphate buffer, e.g., potassiumphosphate buffer, hemin, L-cystine, and optionally a disinfectantneutralizer. In particular embodiments of the first aspect of theinvention, the composition consists of casein digest, soybean digest,animal tissue digest, yeast extract, dextrose, a phosphate buffer, e.g.,potassium phosphate buffer, hemin, L-cystine, a gelling agent, andoptionally a disinfectant neutralizer. In particular embodiments thesecond aspect of the invention, the composition consists of caseindigest, soybean digest, animal tissue digest, yeast extract, dextrose, aphosphate buffer, e.g., potassium phosphate butter, hemin, L-cystine,purified water, and optionally a disinfectant neutralizer. In otherembodiments of the second aspect of the invention, the compositionconsists of casein digest, soybean digest, animal tissue digest, yeastextract, dextrose, a phosphate buffer, e.g., potassium phosphate buffer,hemin, L-cystine, purified water, sheep blood (e.g., defibrinated sheepblood or laked sheep blood), and optionally a disinfectant neutralizer.In other embodiments of the second aspect of the invention, thecomposition consists of casein digest, soybean digest, animal tissuedigest, yeast extract, dextrose, a phosphate buffer, e.g., potassiumphosphate butter, hemin, L-cystine, purified water, sheep blood (e.g.,defibrinated sheep blood or laked sheep blood), a surfactant (e.g.,polysorbate 20), and optionally a disinfectant neutralizer. In certainembodiments of the third aspect of the invention, the compositionconsists of casein digest, soybean digest, animal tissue digest, yeastextract, dextrose, a phosphate buffer, e.g., potassium phosphate buffer,hemin, L-cystine, purified water, a gelling agent, and optionally adisinfectant neutralizer. In other embodiments of the third aspect ofthe invention, the composition consists of casein digest, soybeandigest, animal tissue digest, yeast extract, dextrose, a phosphatebuffer, e.g., potassium phosphate buffer, hemin, L-cystine, purifiedwater, sheep blood (e.g., defibrinated sheep blood or laked sheepblood), a gelling agent, and optionally a disinfectant neutralizer. Inother embodiments of the third aspect of the invention, the compositionconsists of casein digest, soybean digest, animal tissue digest, yeastextract, dextrose, a phosphate buffer, e.g., potassium phosphate buffer,hemin, L-cystine, purified water, sheep blood (e.g., defibrinated sheepblood or laked sheep blood), a surfactant (e.g., polysorbate 20), agelling agent, and optionally a disinfectant neutralizer, In any ofthese embodiments, the disinfectant neutralizer may be omitted. In anyof these embodiments, the amounts of the components may be as shown inTable 1 or 2.

The composition of any aspect of the Invention may also include a dye orstain, in particular, a stain for live cells. e.g.,5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (also known as X-Gal;this reagent is an indicator of the presence of a β-lactamase enzyme).This reagent may be used in conjunction with tetrazolium salts (e.g.,nitroblue letrazollum or tetrazolium red). Other dyes that may be usedin the compositions of any aspect of the invention include Salmon-Gal,Magenta-Gal, and Green-Gal.

The term “about,” as used herein, refers to a value that is ±10% of therecited value.

The term “buffer capacity.” as used herein, refers to the number ofmilimoles of a strong monoprotic acid or a strong monobasic baserequired to alter the pH of a liquid or a gel composition by 1.0.

The units “g/kg,” “mol/kg,” and “mL/kg” indicate the ratio of the amountof the ingredient to the total mass of the composition.

The term “purified water,” as used herein, refers to water that meets orexceeds the standards for purified water set forth in the United StatesPharmacopeia and National Formulary (USF 37-NF32), monograph <1231>,2014.

A composition that is “substantially free of sodium” contains less than50 g/kg of sodium ions, e.g., Jess than 10 g/kg or less than 1 g/kg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A Is a graph of recoveries of bleach-stressed spores of B.subtilis in Growth Direct™ Sterility cassettes with different mediamodifications. The bars labeled (1) represent the data for Schaedlerbroth prepared according to the original recipe. The bars labeled (2)represent the data for a growth medium prepared according to theoriginal recipe for Schaedler broth but without addition oftris(hydroxymethyl)aminomethane. The bars labeled (3) represent the datafor a growth medium prepared according to the original recipe forSchaedler broth but without addition of sodium chloride. The barslabeled (4) represent the data for a growth medium prepared according tothe original recipe for Schaedler broth but with addition of a potassiumsource. The bars labeled (5) represent the data for the growth media ofthe invention. The bars labeled (6) represent the data for Schaedlerchocolate agar medium, The data were normalized to the observedrecoveries on Schaedler chocolate agar.

FIG. 1B is a graph of recoveries of bleach-stressed spores of B.subtilis in Growth Direct™ Sterility cassettes. The bar labeled (1)represents the data for Schaedler blood broth. The bar labeled (2)represents the data for the growth media of the invention. The growthmedia were prepared win a new lots of the individual ingredients. Thedata were normalized to the observed recoveries on Schaedler chocolateagar.

FIG. 2 is a graph of recoveries of Methylobacterium radiotolerans inGrowth Direct™ Sterility cassettes. The bar labeled (1) represents thedata for Schaedler blood broth. The bar labeled (2) represents the datafor the growth media of the invention. The data for TSA on Petri platesis labeled (3). The data could not be normalized to the recovery of M.radiotolerans from trypticase soy agar on Petri plates, because nogrowth of M. adiotolerans was observed on this medium.

FIG. 3 is a graph of recoveries of nine different microorganisms grownon liquid media. The bars labeled (a) represent the data for TSB. Thebars labeled (b) represent the data for Schaedler blood broth. The barslabeled (c) represent the data for the growth media of the invention.The data were normalized to the observed recoveries on the growth mediaof the invention. The 70% cutoff is shown to indicate inferior growth,as compared to the growth media of the invention. The 142% cutoff isshown to indicate superior growth, as compared to the growth media ofthe invention. The 142% cutoff was selected, as the ratio of 100% to142% gives 70%.

FIG. 4 is a graph of recoveries of ten different stressed microorganismsgrown on the growth media of the invention relative to the recoveries ofthe same on TSA. The stress sources are identified in the figure asbleach, heat, spor-klenz® (mixture of hydrogen peroxide, peracetic acid,and acetic acid), thimerosal, and nutrient. The recoveries on the growthmedium of the invention are shown in percentages of the recoveries onTSA of the corresponding stressed microorganisms.

FIG. 5 is a graph showing recoveries of anaerobes C sporogenes and P.acnes on the growth medium of the invention (a), as determined byenumeration o colonies, vs. MPN analysis from FTM media.

FIG. 6 is a graph showing recoveries of bleach-stressed B. subtilis, S.japonica, and D. nishinomioyaensis on the growth medium of the inventionwith (left bar) or without (middle bar) polysorbate 20. The data for thegrowth medium of the invention are compared to the recoveries of thesame microorganisms on TSA.

DETAILED DESCRIPTION

Growth Media Composition:

The invention provides an improved growth medium that promotes thatgrowth of a wide variety of organisms from many genera, includinghuman-associated organisms, anaerobes, water organisms, environmentalorganisms, and molds. Accordingly, the medium may be used as a generalpurpose medium for assays and cell culture. The versatility of themedium allows it to be used in lieu of trypticase. soy broth (TSB) andfluid thioglycollate medium (FTM) in a sterility test. Exemplary fieldsof use for the growth media of the invention include general cellculture and testing liquid, air, soil, surfaces, industrial or clinicalsamples, pharmaceutical products (sterile or non-sterile), foodproducts, beverage products, of nutritional supplements for microbialbioburden. The growth medium may also be employed in other assays, suchas clinical assays, e.g., for blood or other infections, and assays forannbionic resistance. The invention features a composition containingcasein digest, soybean digest, animal tissue digest, yeast extract,dextrose, phosphate buffer, hemin, and L-cystine. The composition may bea solid, which may be mixed with a liquid (e.g., water and/or sheepblood (e.g., defibrinated sheep blood or laked sheep blood)) to preparethe growth media of the invention according to the methods of theinvention. Compositions may also include a surfactant. Preferably, thecomposition does not contain tris(hydroxymethyl)aminonethane and/oradded sodium. The composition is not sodium-free, as thebiologically-derived ingredients may contain sodium. This composition ofthe invention is advantageous as low-sodium content provides superiorgrowth of oxidatively-stressed B. subtilis, as compared to the growth ofthe same on Schaedler broth. In addition, higher potassium content,e.g., from use of potassium phosphate buffer rather than Tris, led tosuperior growth of, e.g., oxidatively-stressed B. subtilis, as comparedto the growth of the same on Schaedler broth.

One of skill in the art can establish the final proportions of one ormore of potassium phosphate or a hydrate thereof, potassium hydrogenphosphate or a hydrate thereof, and potassium dihydrogen phosphate or ahydrate thereof in the phosphate buffer through routine calculations,e.g., by using the desired pH value and aqueous pKs values for therelevant conjugate acids in the Henderson-Hasselbalch equation, A pH ofabout 7.3 (e.g., 7.3±0.5, such as 7.3±0.2) is desired for the media ofthe invention.

A non-limiting example of a composition of the invention is provided inTable 3.

TABLE 3 Ingredient Quantity casein digest 5.6 g soybean digest 1 g yeastextract 5 g animal tissue digest 5 g dextrose 5.8 g dipotassiumphosphate 2.5 g monopotassium phosphate 0.31 g L-cystine 0.4 g hemin0.01 g

This composition in Table 3 may also contain from about 12 to about 15 gof agar (e.g., about 13.5 g of agar). This composition may further bedissolved or suspended in about 1 L of aqueous medium, e.g. about 950 mLof purified water and about 50 mL of sheep blood (e.g., defibrinatedsheep blood or laked sheep blood). The composition may further contain0.05% (w/V) of polysorbate 20. In a non-limiting example, thecomposition described in Table 3 is combined with 950 mL of purifiedwater, which can further include 50 mL of sheep blood (e.g., laked), and10 mL of 5% (w/v) polysorbate 20 solution can be added.

Solid compositions of the invention contain casein digest, soybeandigest, animal tissue digest, yeast extract, dextrose, a phosphatebuffer, hemin, and L-cystine. Certain solid compositions of theinvention contain less than about 60 g/kg (e.g., less than about 55 g/kgor less than about 50 g/kg) of sodium ions, Solid compositions of theinvention may also contain a gelling agent. A solid composition of theinvention that includes a gelling agent affords a gel upon mixing withwater, sheep blood, or both. Non-limiting examples of gelling agentsinclude agar, gellan, sodium alginate, xanthan gum, guar gum, gelatin,agarose, Eladium™ (a polysaccharide produced by Rhizobium sp. (CNCMnumber: 1-1809)), and combinations thereof. One of skill in the art candetermine the quantity of a liquid (e.g., purified water and/or sheepblood) that is required for the preparation of a gel suitable for use asa growth medium. The amount of the gelling agent in the solidcomposition of the invention depends on the identity of the gellingagent. For example, when the gelling agent present in the dry solid ofthe invention is gellan, its concentration can be between about 50 g/kgand about 300 g/kg, and preferably between about 130 g/kg and about 230g/kg (e.g., about 180 g/kg). When the gelling agent present in the gelor liquid of the invention is gellan, its concentration can be betweenabout 2.5 g/kg and about 13 g/kg (e.g., about 6.8 g/kg), In anotherexample, when the gelling agent present in the dry solid of theinvention is xanthan gum or sodium alginate, Its concentration can bebetween about 70 g/kg and about 400 g/kg, and preferably between about160 g/kg and about 300 g/kg (e.g., about 220 g/kg). When the gellingagent present in the gel of the invention is xanthan gum or sodiumalginate, its concentration can be between about 3.4 g/kg and about 13g/kg (e.g., about 6.8 g/kg). In yet another example, when the gellingagent present in the gel of the invention is polyacrylamide. Itsconcentration can be between about 50 g/kg and about 200 g/kg (e.g.,about 150 g/kg). In certain examples, when the gelling agent present inthe dry solid of the invention is guar gum, its concentration can bebetween about 400 g/kg and about 800 g/kg (e.g., about 650 g/kg). Whenthe gelling agent present in the gel of the invention is guar gum, itsconcentration can be between about 10 g/kg and about 40 g/kg (8.9.,about 21 g/kg).

Liquid compositions of the invention contain purified water or sheepblood or both. Liquid compositions may also include a gelling agent at aconcentration that is too low to form a gel. The concentrations ofingredients in liquid compositions of the invention are as describedherein.

Casein digest used in the compositions of the invention can be preparedaccording to methods known in the art by hydrolysis of casein proteinfrom bovine milk. Soybean digest used in the compositions of theinvention can be prepared according to methods known in the art, e.g.,through enzymatic digestion of defatted soy flour that was heat-treatedto remove heat-labile protease inhibitors. Animal tissue digest used inthe compositions of the invention can be prepared according to methodsknown in the art, e.g.; through hydrolysis of meat from muscle tissue oroffal and gelatin. Yeast extract is defined in the USP as “awater-soluble, peptone-like derivative of yeast cells ([e.g.,]Saccharomyces)” and is readily available as a spray-dried powder.Commercially available casein digest: soybean digest, animal tissuedigest, and yeast extract may be used in the compositions of theinvention. These ingredients may be obtained, e.g., from BD Biosciences(San Jose, CA), Each of these ingredients contains less than 15% (w/w)of sodium,

Defibrinated sheep blood can be prepared according to methods known inthe art by aseptic collection of blood from sheep and subsequentmechanical removal of fibrin during the clotting process of thecollected blood in the absence of anticoagulants. Commercially availabledefibrinated sheep blood (e.g., from Rockland Immunochemicals Inc.,Gilbertsville, PA) may be used in the compositions of the invention.Sheep blood is known to contain sodium (up to 3.48+0.02 g/100 mL; see,e.g., Long et al., J. Anim. Sci, 24:145-150, 1965), Laked sheep bloodcan be prepared by hemolysis of the defibrinated sheep blood.Commercially available laked sheep blood can be used in the compositionsof the invention (e.g., from Cedarlane, Burlington, NC).

Surfactants (e.g., nonionic surfactants) can be used in compositions ofthe invention to control sediment formation in liquid and gelcompositions of the invention. The surfactants can be a Poloxamer, aPolysorbate, or a Triton. These surfactants are commercially availablefrom various chemical suppliers, such as Dow Chemical, Midland, MI, andSigma Aldrich, St. Louis, MO, A preferred surfactant is polysorbate 20.

The growth media of the invention may also include an antibiotic, as isknown in the art. The growth medium may further include a disinfectantneutralizer. Examples of disinfectants to be neutralized includealcohols, hypochlorite, hydrogen peroxide, acetic acid, peroxyaceticacid, quaternary ammonium compounds, phenolics, iodine, chlorinepreparations, mercurials, formaldehyde, and glutaraldehyde. Examples ofneutralizers include histidine, thiosulfate, polysorbate 80, and/orlecithin. Other neutralizers includes bisulfite, glycine, divalentcations (e.g., Mg²⁺ or C²⁺), and thioglycollate. As described above, thecompositions of the invention preferably include non-blologicallyderived ingredients that are substantially free of sodium, i.e., thecompositions of the invention contain less sodium than Schaedler broth.Preferably, the compositions of the invention do not containtri(hydroxymethyl)aminomethans (Tris). Absence of this ingredient isadvantageous, as quality of Tris is subject to lot-to-lot variationsthat may lead to poor reproducibility of recoveries of microorganismsgrown on media containing this ingredient. A phosphate buffer, e.g. apotassium phosphate buffer, is therefore used in the compositions of theinvention.

Methods of Culturing a Population of Cells

The growth media of the invention may be employed as a general growthmedium for assays and cell culture. The use of the growth media of theinvention is particularly a antageous in growth based sterility assays,e.g., involving the use of cell culture devices employing permeablemembranes for growth of microorganisms (e.g., bacteria or fungi). Suchcell culture devices are described in detail in InternationalPublication Nos. WO 2007/038478, WO 2013/070730, and WO 2013/158666, thedisclosures of which are incorporated herein by reference in theirentirety. In particular, the growth media of the invention may be usedwith a Growth Direct Sterility cassette according to the methodsdescribed in WO 2013/070730. The particular advantages include rapidachievement of reliable sterility test results, thereby allowing forefficient cost control in healthcare and manufacturing.

In particular, the growth media of the invention can be employed in ananalog of the compendial test that employs TSA and FTM. For example, themedium can be employed in a set of three assays, one for aerobesincubated at 32.5° C., one for anaerobes incubated at 32.5° C., and oneof aerobes incubated at 22° C. Other uses of the growth media includeenvironmental monitoring, bioburden testing, clinical and diagnosticuses, antibiotic resistance testing, and antibiotic selection. Fortesting antibiotic susceptibility or selecting cells having antibioticresistance (e.g., after translection), the growth media may include anantibiotic, as is known in the art.

The use of the growth media of the invention is not limited to thesettings involving growth of microorganisms on permeable membranes. Forexample, the growth media of the invention may be used to culturemicroorganisms (e.g., bacteria or fungi) in test tubes, Petri plates,Rodac plates, microfluidic cell culture devices (such as those describedin, e.g., U.S. Patent Applica cation Publication Nos. 2013/0090268 and2013/0171679), bioreactors (such as Eppendorf CellGene bioreactors orthose described in, e.g., U.S. Patent Application Publication Nos.2013/0196375 and 2014/0024105), and other cell culture vessels.

Samples that can be assayed using the growth media of the invention arenot limited and include industrial samples (e.g., raw, in-process, orfinished material in the manufacture of foods, beverages, or nutritionalsupplements; raw, In-process, or finished material in the manufacture ofmedical or in vitro diagnostic devices; chemical products; Industrialsurfaces; instrumentation; or machinery), pharmaceuticals and reagentused in preparing pharmaceuticals (e.g., raw, finished, or in-processmaterial in the manufacture of pharmacological, cosmetic, blood, orother products for topical or internal use in humans or animals),biological samples, environmental samples (e.g., water samples (such asnatural bodies of water (such as rivers, lakes, ponds, and oceans),waste water, and treated sources of water (such as municipal watersupplies)), air samples, soil samples, and surface samples). Surfacesthat may be tested include equipment, materials, and facilities used inthe manufacture, packaging, of storage of goods (e.g., pharmaceuticals); equipment, materials, and facilities used in research; clothing.bedding, and other fabrics (e.g., for medical providers or patients),and equipment, materials, and facilities used in treatment (e.g.,hospitals, clinics, and doctor's offices).

The non-limiting examples of the genera of microorganisms (e.g. bacteriaor fungi) that can be cultured. using the growth media of the inventioninclude Acinetobacter (e.g. Acinetobacter lwofii). Aspergillus (e.g.Aspergillus brasiliensis or Aspergillus fumigates), Bacillus (e.g.Bacillus clausil, Bacillus idriensis, Bacillus licheniformis, orBacillus substilis). Candida (e.g., Candida albicans), Clostridium(e.g., Clostridum sporogenes). Corynebacterium(e.g., Corynebacteriumtuberculostearicum or Corynebacterium xerosis), Dermacoccus(e.g.Dermacoccus nishinomlyaensis), Escherichia(e.g., Escherichia coli),Exserohilum (e.g., Exserohilum rostratum), Kocuria(e.g., Kocuriarhizophila), Methylobacterium (e.g., Methylobacterium radiotolerans),(e.g., Micrococcus luteus), Paenibacillus (e.g., Paenibacillusglucanolyticus), Penicillium(e.g., Penicillium chrysogenum orPenicillium notatum), Pseudomonas (e.g., Pseudomonas aeruginosa orPseudomonas fluorescens), Sphingomonas (e.g., Sphingomonas japonica),Staphylococcus (e.g., Straphylococcus aureus, Staphylococcusepidermidis, or Staphylococcus hominis), Streptococcus (e.g.,Streptococcus pyogenes). and Streptomyces (e.g., Streptomyceshalstedii). In particular, the growth media of the invention allow forreproducibly good recovery of numerous microorganisms, includingbleach-stressed B. subtilis.

Kits of the Invention

The invention also features kits containing the compositions of theinvention described above. The compositions of the invention may beincluded in the kits of the invention as dry goods, e.g., powders. gels,or liquids. The solids, e.g. powders, may be packaged as a mixture in asingle container (e.g. a bottle, an ampoule, or a jar). Alternatively,the solids, e.g., powder, may be packaged in separate containers (e.g.,a bag, a can, a pouch, a bottle, a vial, an ampoule, a jar, or acombination thereof). The mixture may contain one or more of caseindigest, soybean digest, animal tissue digest, yeast extract, dextrose, aphosphate buffer, hemin, L-cystine, and a gelling agent (e.g., agar,gellan, sodium alginate, xanthan gum, guar gum, gelatin, agarose,Eladium™, or a combination thereof). The relative quantities of theseingredients are as described above.

The following examples are meant to Illustrate the invention and are notmeant to limit the invention in any way.

EXAMPLES Example 1: Preparation of growth media of the invention

A composition containing purified water (950 ml.), casein digest (6.6 g.Neogen or BD Biosciences): soybean digest (1 g. Neogen), dipotassiumphosphate (2.6 g. Sigma-Aldrich), dextrose (5.82 g. Sigma-Aldrich),animal tissue digest (5 g, BD Biosciences or Neogen), yeast extract (5g, BD Biosciences or Neogen), monopotassium phosphate (0.31 g,Sigma-Aldrich), hemin (0.01 g, Sigma-Aldrich), and L-cystine (0.4 g,Sigma-Aldrich) was autoclaved. The composition was cooled to roomtemperature, and pH was adjusted to 7.3 (±0.2) using aqueous KOH or HCl.The composition was then heated to 65° C. and 50 ml of defibrinatedsheep blood (Northeast Laboratories or Thermo Scientific) was added. Thetemperature was held constant until the color changed from red to brown,at which time the composition was allowed to cool to ambient temperature(room temperature, about 22° C.

Example 2: Comparison of growth of microorganisms on various growthmedia

This example illustrates the versatility of the growth media of theinvention in supporting the growth of various microorganisms as comparedto the standard media used in the growth-based sterility assaysdescribed in United States Pharmacopeia monograph <71>. The standardmedia are TSB and FTM.

The growth media of the invention were tested against a variety oforganisms. The media of the invention were also compared to other growthmedia known in the art. All tests were carried out in Growth DirectSterility cassettes (Rapid Micro Biosystems, Bedford, MA) or Petriplates.

Comparison of modifications of Schaedler broth to the original recipe,Schaedler chocolate agar, and growth medium of the invention

The recovery of bleach-stressed B. subtilis on Schaedler broth preparedaccording to the original recipe (1). Schaedler broth without Tris (2),Schaedler broth containing non-biologically-derived ingredients that aresubstantially free of sodium (3), Schaedler broth that is high inpotassium (4), the growth medium of the invention (5), and Schaedlerchocolate agar (6) are shown in FIG. 1A. The tests were performed intriplicate with two different batches of each medium. The results werenormalized to the recovery on Schaedler chocolate Medium. As shown inFIG. 18 , the results are reproducible, even with different underlyinglots of the powdered ingredients and/or blood.

Comparison of the recovery using growth media of the invention in GrowthDirect™ Sterility cassettes to the recovery using the TSA on Petriplates

The growth of 23 different organisms on growth media of the invention inGrowth Direct™ Sterility cassettes was compared to the growth of thesame organisms on TSB on Petri plates. The results were normalized tothe observed growth on TSA on Petri plates. Only three organismsexhibited recovery below the 70% cutoff (the USP standard) for goodgrowth. For two organisms, Dermacoccus nishinomiyaensis and M.radiotolerans, growth in the Sterility Cassette on the growth media ofthe invention was consistently superior. FIG. 2 shows the dataMethylobacterium radiotolerans; however, the growth could not benormalized to TSA because no growth was observed on TSA.

Comparison of growth of microorganisms in Growth Direct™ Sterilitycassettes using TSB, Schaedler blood broth, and growth media of theinvention

Growth on different media within the context of the Sterility Cassettewas investigated to eliminate the variable of agar. The focus of thisstudy was on problematic organisms. FIG. 3 shows this comparison, withthe data normalized to growth on the growth media of the invention. Forlive organisms, there is no significant difference among all the media.All growth falls between the 70% and 142% cutoffs. For two organisms, C.xerosis and Exserohilum rostratum, growth is superior on the growthmedia of the invention. For S. halstedii and P. chrysogenum, growth onTSB is superior, yet in all cases, growth is detected. No organism wasidentified that can grow on TSA, TSB, or Schaedler blood or chocolateagar, that cannot grow on growth media of the invention.

Comparison of growth of microorganisms on growth media of the inventionto the growth of the same microorganisms on FTM

FTM is designed for growing anaerobes in a liquid format. The media hasboth aerobic and anaerobic layers, so it is also meant to be anall-purpose media. Because it is a liquid growth media but does notsupport growth on the surface of a filtration membrane, growth can onlybe monitored by the presence of turbidity. The presence/absence natureof this information can be converted to quantitative data using a MostProbable Number (MPN) method, where replicate 10-fold dilutions of lowlevel inocula are monitored for growth, and the pattern ofgrowth/no-growth can be converted to a most probable inoculum for themost concentrated inoculation.

This method was used to compare the growth of Propionibacterium acnes inanaerobic sterility cassettes with the growth media of the invention togrowth in FTM using MPN method. As shown in Table 4, counts in theanaerobic cassette are similar to those in FTM using MPN method.However, P. acnes grew significantly faster on the growth media of theinvention compared to growth in FTM.

TABLE 4 Recovery of Propionibacterium acnes in Growth Direct ™ cassettesand FTM. FTM Anaerobic sterility cassette with (Most Probable growthmedia of the invention Number analysis) Count 28.2 23 Days to detection<5 days by eye >7 days by eye

The growth of some aerobic organisms in FTM was compared to their growthon the growth media of the invention. The focus of this test was onmolds, the recoveries of which were observed to be below 70% in GrowthDirect™ sterility cassettes with the growth media of the invention whencompared to growth on TSA. MEN analysis was performed to obtainquantitative data from the growth/no growth information of FTM. As shownin Table 5, the molds grew very poorly in FTM as compared to theirgrowth on TSA of even Schaedler chocolate agar (SCA) Growth in sterilitycassettes on media of the invention was significantly better. (Compare6^(th) column, % growth in FTM, to last column, % growth in cassettes.Growth was normalized to TSA controls.)

TABLE 5 Recovery of molds TSA, SCA, FTM, and the growth media of theinvention 32.5° C. FTM 22.5° C. 22.5° C. TSA SCA FTM MPN % TSA TSA SCATSB Cassette A. brasiliensis 17.5 11.5 1/3 0.41 2% 17.5 13.0 3/3 76% A.fumigates 17.0 11.5 2/3 1.1 6% 5.5 8.0 3/3 68% P. chrysogenum 10.5 7.50/3 <0.41 <4%  4.5 10.0 3/3 37%

Example 3

A growth medium composition containing purified water (950 mL), caseindigest (5.6 g), soybean digest (1 g), dipotassium phosphate (2.5 g),dextrose (6.82 g), animal tissue digest (5 g), yeast extract (5 g),monopotassium phosphate (0.31 g), hemin (0.01 g), L-cystine (0.4 g), andlaked sheep blood (50 ml.) or defibrinated sheep blood (50 ml) having apH of 7.3±0.2 was employed. The composition containing laked sheep bloodwas prepared as described in Example 1 with the exception that lakedsheep blood was added at 45° C. or cooler to the mixture prepared asdescribed in Example 1.

Membrane filters were placed on broth soaked pads. The growth promotionof the composition was compared to trypticase soy agar (TSA) using asuite of test organisms inclusive of human-associated strains, waterorganisms, yeast, USP microorganisms, mold and a number of spore-formingBacillus sp. (Table 6). In Table 6. % recovery indicates the recovery ofmicroorganisms on the growth medium of the invention (not containingpolysorbate 20) as a percentage of recovery on TSA.

TABLE 6 Laked or defibrinated blood Organism % Recovery SoilMicroorganisms defibrinated blood Acinetobacter lwoffii 121 defibrinatedblood Paenibacillus glucanolyticus 79 defibrinated blood Streptomyceshalstedii 71 defibrinated blood Bacillus clausii 106 defibrinated bloodBacillus licheniformis 100 Mold Spores defibrinated blood Penicilliumnotatum 83 laked blood Penicillium chrysogenum 86 Water Microorganismslaked blood Methylobacterium radiotolerans 213 laked blood Sphingomonasjaponica 613 laked blood Pseudomonas fluorescens 98 USP Microorganismslaked blood Escherichia coli 88 laked blood Staphylococcus aureus 84laked blood Pseudomonas aeruginosa 116 laked blood Bacillus subtilis 158laked blood Candida albicans 113 laked blood Aspergillus brasiliensis 92Human-associated Microorganisms defibrinated blood Staphylococcusepidermidis 92 defibrinated blood Staphylococcus warnerii 96defibrinated blood Staphylococcus hominis 104 defibrinated bloodStaphylococcus capitis 177 laked blood Staphylococcus haemolyticus 115laked blood Corynebacterium xerosis 6550 defibrinated blood Kocuriarhizophila 106 laked blood Dermacocous nishinomiyaensis 98 defibrinatedblood Corynebacterium 101 tuberculostearicum defibrinated bloodMicrococcus luteus 110 laked blood Streptococcus pyogenes 94

Further, recoveries of stressed microorganisms on the growth medium ofthe invention were compared to the recoveries on TSA by following theprocedure described above. As shown in FIG. 4 , recoveries of allbleach-stressed, heat-stressed, and nutrient-stressed microorganismswere superior on the growth. medium of the invention as compared to TSA.Recoveries of spor-klenz®-stressed and thimerosal-stressedmicroorganisms on the growth medium of the invention were comparable tothose on TSA. Growth medium of the invention used in this test wasprepared with defibrinated sheep blood and without polysorbate 20.

In all cases, the growth medium of the invention exhibited growthpromotion that was comparable or better than TSA across all the strainstested. In no case was recovery of the test strains on the growth mediumof the invention substantially inferior to that of TSA.

Additionally, growth of the medium of the invention was compared withcalculated MPN from organisms spiked and grown in FTM as described bythe compendial sterility test. The anaerobic organisms P. acnes and C.sporogenes were tested to see if the growth medium of the invention, wunder anaerobic conditions, exhibited the same growth promotion as FTM.As shown, both strains exhibited comparable recovery on membrane filtersincubated anaerobically on cellulose pads with the growth medium of theinvention vs. the MPN obtained from incubation in FTM (FIG. 5 ).

Example 4

A growth medium composition containing purified water (950 mL), caseindigest (5.6 g), soybean digest (1 g), dipotassium phosphate (2.5 g),dextrose (5.82 g), animal tissue digest (5 g), yeast extract (5 g),monopotassium phosphate (0.31 g), hemin (0.01 g), L-cystine (0.4 g),laked sheep blood (50 mL), and 10 mL of 5% (w/v) polysorbate 20 having apH of 7.3±0.2 was employed. The composition was prepared as described inExample 1 with the exception that laked sheep blood was added at 45° C.or cooler to the mixture prepared as described in Example 1.

Growth of bleach-stressed B. subtilis. S. japonica, and D.nishinomiyaensis on the media of the invention containing polysorbate 20was compared to the growth on the media of the invention free ofpolysorbate 20 and to the growth on TSA. The results are shown in FIG. 6. The growth medium of the invention containing polysorbate 20 was alsotested for its capability of supporting the growth of anaerobes (e.g.,P. acnes). P. acnes were grown on Growth Direct™ anaerobic sterilitycassettes using the growth medium of the invention and the recovery wascompared to growth on blood agar plates in BD anaerobic GasPak™ pouches.Recovery of P. acnes on the growth medium of the invention was 13.2 CFUon average as compared to 18 CFU for blood agar control.

The invention is also described by the following numbered embodiments.

1. A composition comprising casein digest, soybean digest, animal tissuedigest, yeast extract, dextrose, a phosphate buffer, hemin, andL-cystine.

2. The composition of embodiment 1, wherein, at 22° C., said compositionis solid.

3. The composition of embodiment 2, wherein said composition is apowder.

4. The composition of embodiment 2 or 3, wherein, excluding saidphosphate buffer from the total mass of said composition, saidcomposition comprises bet and about 500 g/kg of casein digest.

5. The composition of embodiment 4, wherein, excluding said phospriatebuffer from the total mass of said composition, said compositioncomprises between about 60 g/kg and about 400 g/kg of casein digest.

6. The composition of embodiment S, wherein, excluding said phosphatebuffer from the total mass of said composition, said composition compween about 100 g/kg and about 300 g/kg of casein digest.

7. The composition of embodiment 6, wherein, excluding said phosphatebuffer from the total mass of said composition, said compositioncomprises about 245.3 g/kg of casein digest.

8. The composition of any one of embodiments 2 to 7, wherein, excludingsaid phosphate buffer from the total mass of said composition, saidcomposition comprises between about 0.5 g/kg and about 300 g/kg ofsoybean digest.

9. The composition embodiment 8, wherein said, excluding said phosphatebuffer from the total mass of said composition, comprises between about10 g/kg and about 200 g/kg of soybean digest.

10. The composition of embodiment 9, wherein, excluding said phosphatebuffer from the total mass of said composition, said compositioncomprises between about 10 g/kg about 100 g/kg of soybean digest.

11. The composition of embodiment 10, wherein, excluding said phosphatebuffer from the total mass of said composition, said compositioncomprises about 43.8 g/kg of soybean digest.

12. The composition of any one of embodiments 2 to 11, wherein,excluding said phosphate buffer from the total mass of said composition,said composition comprises between about 1 g/kg and about 500 g/kg ofanimal tissue digest.

13. The composition of embodiment 12, wherein, excluding said phosphatebuffer from the total mass of said composition, said compositioncomprises between about 50 g/kg and about 400 g/kg of animal tissuedigest.

14. The composition of embodiment 13, wherein, excluding said phosphatebuffer from the total mass of said composition, said compositioncomprises between about 100 g/kg and about 300 g/kg of animal tissuedigest.

15. The composition of embodiment 14, wherein, excluding said phosphatebuffer from the total mass of said composition, said compositioncomprises about 219 g/kg of animal tissue digest.

16. The composition of any one of embodiments 2 to 15, wherein,excluding said prosphate buffer from the total mass of said composition,said composition comprises between about 1 g/kg and about 500 g/kg ofyeast extract.

17. The composition of embodiment 16, wherein, excluding said phosphatebuffer from the total mass of said composition, said compositioncomprises between about 50 g/kg and about 400 g/kg of yeast extract.

18. The composition of embodiment 17, wherein, excluding said phosphatebuffer from the total mass of said composition, said compositioncomprises between about 100 g/kg and about 300 g/kg of yeast extract.

19. The composition of embodiment 18, wherein, excluding said phosphatebuffer from the total mass of said composition, said compositioncomprises about 219 g/kg of yeast extract.

20. The composition of any one of embodiments 2 to 19, wherein,excluding said phosphate. buffer from the total mass of saidcomposition, said composition comprises between about 1 g/kg and about500 g/kg of dextrose.

21. The composition of embodiment 20, wherein, excluding said phosphatebuffer from the total mass of said composition, said compositioncomprises between about 50 g/kg and about 400 g/kg of dextrose.

22. The composition of embodiment 21, wherein, excluding said phosphatebuffer from the total mass of said composition, said compositioncomprises between about 100 g/kg and about 300 g/kg of dextrose.

23. The composition of embodiment 22 wherein, excluding said phosphatebuffer from the total mass of said composition, said compositioncomprises about 258 g/kg of dextrose.

24. The composition of any one of embodiments 2 to 23, wherein saidcomposition comprises a quantity of phosphate buffer sufficient toprovide a buffer capacity of from 0.1 mmol/(pH unit) to 100 mmol/(pHunit) upon dissolution in an aqueous medium.

25. The composition of embodiment 24, wherein said composition comprisesa quantity of phosphate buffer sufficient to provide a buffer capacityof from 1 mmol/(pH unit) to 50 mmol/(pH unit) upon dissolution in anaqueous medium.

26. The composition of embodiment 25, wherein said composition comprisesa quantity of phosphate buffer sufficient to provide a buffer capacityof from 2 mmol/(pH unit) to 20 mmol/(pH unit) upon dissolution in anaqueous medium.

27. The composition of embodiment 26, wherein said composition comprisesa quantity of phosphate buffer sufficient to provide a buffer capacityof from 3 mmol/(pH unit) to 10 mmol/(pH unit) upon dissolution in anaqueous medium.

28. The composition of any one of embodiments 2 to 27, wherein saidcomposition comprises between about 0.2 g/kg and about 1 g/kg of hemin.

29. The composition of embodiment 28, wherein said composition comprisesabout 0.4 g/kg of hemin.

30. The composition of any one of embodiments 2 to 29, wherein,excluding said phosphate buffer from the total mass of said composition,said compo comprises between about 8 g/kg and about 40 g/kg ofL-cystine.

31. The composition of embodiment 30, wherein, excluding said phosphatebuffer from the total mass of said composition, said compositioncomprises between about 12 g/kg and about 20 g/kg of L-cystine.

32. The composition of embodiment 31, wherein, excluding said phosphatebuffer from the total mass of said composition, said compositioncomprises about 17.5 g/kg of L-cystine.

33. The composition of any one of embodiments 2 to 32, wherein saidcomposition further comprises a gelling agent.

34. The composition of embodiment 33, wherein said gelling agent isselected from the group consisting of agar, gellan, sodium alginate,xanthan gum, guar gum, gelatin, agarose, and a polysaccharide producedby Rhizobium sp. (CNCM number: 1-1809).

35. The composition of embodiment 34, wherein said gelling agent isagar.

36. The composition of any one of embodiments 33 to 35 wherein saidcomposition comprises between about 10 g/kg and about 800 g/kg of saidgelling agent.

37. The composition of embodiment 36, wherein said composition comprisesbetween about 100 g/kg and about 600 g/kg of said gelling agent.

38. The composition of embodiment 37, wherein said composition comprisesbetween about 250 g/kg and about 450 g/kg of said gelling agent.

39. The composition of embodiment 38, wherein said composition comprisesabout 350 g/kg of said gelling agent.

40. The composition of any one of embodiments 2 to 39, wherein saidcomposition further comprises a surfactant.

41. The composition of embodiment 40, wherein said surfactant is apolysorbate.

42. The composition embodiment 40 or 41, wherein said compositioncomprises between about 0.4 g/kg and about 190 g/kg of said surfactant.

43. The composition of embodiment 42, wherein said composition comprisesbetween about 4 g/kg and about 80 g/kg of said surfactant.

44. The composition of embodiment 43, wherein said composition comprisesbetween about 4 g/kg and about 40 g/kg of said surfactant.

45. The composition of embodiment 44, wherein said composition comprisesabout 20 g/kg of said surfactant.

46. The composition of embodiment 1, wherein, at 22° C., saidcomposition is a liquid or a gel.

47. The composition of embodiment 1 or 46 further comprising purifiedwater.

48. The composition of any one of embodiments 1, 46, and 47, furthercomprising sheep blood.

49. The composition of embodiment 48, wherein the concentration of saidsheep blood in said composition is between about 5 mL/kg and about 200mL/kg.

50. The composition of embodiment 49, wherein the concentration of saidsheep blood in said composition is between about 5 mL/kg about 100mL/kg.

51. The composition embodiment 50, wherein the concentration of saidsheep blood in said composition is about 50 mL/kg.

52. The composition of any one of embodiments 48 to 51, wherein redblood cells in said sheep blood are lysed.

58. The composition of any one of embodiments 48 to 52, wherein saidsheep blood is laked sheep blood.

64. The composition of any one of embodiments 48 to 62, wherein saidsheep blood is defibrinated sheep blood.

55. The composition of any one of embodiments 46 to 54, wherein theconcentration of casein digest in said composition is between about 0.1g/kg and about 50 g/kg.

56. The composition of embodiment 55, wherein the concentration ofcasein digest in said composition is between about 1 g/kg and about 20g/kg.

57. The composition of embodiment 56, wherein the concentration ofcasein digest in said composition is between about 2 g/kg and about 10g/kg.

58. The composition of embodiment 57, wherein the concentration ofcasein digest in said composition is about 5.6 g/kg.

59. The composition of any one of embodiments 46 to 58, wherein theconcentration of soybean digest in said composition is between about0.05 g/kg and about 30 g/kg.

60. The composition of embodiment 59, wherein the concentration ofsoybean digest in said composition is between about 0.1 g/kg and about10 g/kg.

61. The composition of embodiment 60, wherein the concentration ofsoybean digest in said composition is between about 0.2 g/kg and about 3g/kg.

62. The composition of embodiment 61, wherein the concentration ofsoybean digest in said composition is about 1 g/kg.

63. The composition of any one of embodiments 46 to 62, wherein theconcentration of animal tissue digest in said composition is betweenabout 0.1 g/kg and about:50 g/kg.

64. The composition of embodiment 63, wherein the concentration ofanimal tissue digest in said composition is between about 1 g/kg andabout 20 g/kg.

65. The composition of embodiment 64, wherein the centration of animaltissue digest in said composition is between about 2 g/kg and about 10g/kg.

66. The composition of embodiment 65, wherein the concentration ofanimal tissue digest in said composition is about 5 g/kg.

67. The composition of any one of embodiments 46 to 66, wherein theconcentration of yeast extract in said composition is between about 0.1g/kg and about 50 g/kg.

68. The composition of embodiment 67, wherein the concentration of yeastextract in said composition is between about 1 g/kg and about 20 g/kg.

69. The composition of embodiment 68, wherein the concentration of yeastextract in said composition is between about 2 g/kg and about 10 g/kg.

70. The composition of embodiment 69, wherein the concentration of yeastextract in said composition is about 5 g/kg.

71. The composition of any one of embodiments 46 to 70, wherein thecentration of dextrose in said composition is tween about 0.1 g/kg andabout 50 g/kg.

72. The composition of embodiment 71, wherein the concentration ofdextrose in said composition is between about 1 g/kg and about 20 g/kg.

73. The composition of embodiment 72, wherein the concentration ofdextrose in said composition is between about 2 g/kg and about 10 g/kg.

74. The composition o embodiment 73, wherein the concentration ofdextrose in said composition is 5.5 g/kg.

75. The composition of any one of embodiments 46 to 74, wherein saidcomposition comprises a quantity of phosphate buffer sufficient toprovide a buffer capacity of from 0.1 mmol/(pH unit) to 100 mmol/pH.

76. The composition of embodiment 75, wherein said composition comprisesa quantity of phosphate buffer sufficient to provide a buffer capacityof from 1 mmol/(pH unit) to 50 mmol/(pH unit) upon dissolution in anaqueous medium.

77. The composition of embodiment 76, wherein said composition comprisesa quantity of phosphate buffer sufficient to provide a buffer capacityof from 2 mmol/(pH unit) to 20 mmol/(pH unit) upon dissolution in anaqueous medium.

78. The composition of embodiment 77. wherein said composition comprisesa quantity of phosphate buffer sufficient to provide a buffer capacityof from 3 mmol/(pH unit) to 10 mmol/(pH unit) upon dissolution in anaqueous medium.

79. The composition of any one of embodiments 46 to 78, wherein theconcentration of hemin in said composition is about 0.01 g/kg.

80. The composition of any one of embodiments 46 to 79, wherein theconcentration of L-cystine in said composition is between about 0.01g/kg and about 0.5 g/kg.

81. The composition of embodiment 80, wherein the concentration ofL-cystine in said composition is about 0.4 g/kg.

82. The composition of any one of embodiments 46 to 81 furthercomprising a surfactant.

83. The composition of embodiment 82, wherein said surfactant is apolysorbate.

84. The composition of embodiment 83, wherein said composition comprisesbetween about 0.01 g/kg and about 5 g/kg of surfactant.

85. The composition of embodiment 84, wherein said composition comprisesbe ween about 0.1 g/kg and about 2 g/kg of said surfactant.

86. The composition of embodiment 85, wherein said composition comprisesbetween about 0.1 g/kg and about 1 g/kg of said surfactant.

87. The composition of embodiment 86, wherein said composition comprisesabout 0.5 g/kg of said surfactant.

88. The composition of any one of embodiments 46 to 87 furthercomprising a gelling agent.

89. The composition of embodiment 88, wherein said gelling agent isselected from the group consisting of agar, gellan, sodium alginate,xanthan gum, guar gum, gelatin, agarose, polyacrylamide, and apolysaccharide produced by Rhizobium sp. (CNGM number: 1-1809).

90. The composition of embodiment 89, wherein said gelling agent isagar.

91. The composition of embodiment 90, wherein said gelling agent is apolysaccharide produced by Rhizobium sp.

92. The composition of embodiment 90 or 91, wherein the concentration ofsaid gelling agent in said composition is between ab g/kg and about 25g/kg.

93. The composition of embodiment 92, wherein the concentration of saidgelling agent in said composition is about 13.5 g/kg:

94. The composition of embodiment 93, wherein said gelling agent isgellan.

95. The composition of embodiment 94, wherein the concentration ofgellan in said composition is between about 1.0 g/kg and about 13 g/kg.

96. The composition of embodiment 95, wherein the concentration ofgellan in said composition is about 6.8 g/kg.

97. The composition of embodiment 89, wherein said gelling agent isxanthan gum or sodium alginate.

98. The composition of embodiment 97, wherein the concentration of saidgelling agent in said composition is between about 3,4 g/kg and about 17g/kg.

99.The composition of embodiment 98, wherein the concentration of saidgelling agent in said composition is about 9 g/kg.

100. The composition of embodiment 89, wherein said gelling agent ispolyacrylamide.

101. The composition of embodiment 100, wherein the concentration ofpolyacrylamide in said composition is between about 50 g/kg and about200 g/kg.

102. The composition of embodiment 101, wherein the concentration ofpolyacrylamide in said composition is about 150 g/kg.

103. The composition of embodiment 89, wherein said gelling agent isguar gum.

104.The composition of embodiment 103, wherein the concentration of guargum in said composition is between about 10 g/kg and about 40 g/kg.

105. The composition of embodiment 104, wherein the concentration ofguar gum in said composition is about 21 g/kg.

106. The composition of any one of embodiments 46 to 87, wherein saidcomposition is a liquid.

107. The composition of any one of embodiments 46 to 105, wherein saidcomposition is a gel.

108. The composition of any one of embodiments 1 and 46-107, whereinsaid composition has a pH of 73±0.5.

109. The composition of any one of embodiments 1 to 108, wherein saidcomposition does not comprise tris(hydroxymethyl)aminomethane.

110. The composition of any one of embodiments 1 to 109, wherein saidcomposition does not contain added sodium.

111. A method of culturing a population of cells comprising contactingsaid population of cells with the composition of any one of embodiments46 to 110 under conditions supportive of growth of said population ofcells.

112. The method of embodiment 111, wherein said population of cells isdisposed on a first side of a membrane, and a second side of saidmembrane is in contact with the composition of any one of embodiments 46to 110.

113. The method of embodiment 112, wherein said membrane is permeable.

114 The method of any one of embodiments 111 to 113, wherein one or morecells within said population of cells are aerobes.

115. The method of any one of embodiments 111 to 113, wherein one ormore cells within said population of cells are anaerobes.

116. The method of embodiment 115, wherein said anaerobe is an obligateanaerobe.

117. The method of any one of embodiments 111 to 113, wherein one ormore cells within said population of cells belong to a genus selectedfrom the group consisting of Acinetobacter, Aspergillus, Bacillus,Corynebacterium, Dermacoccus, Escherichia, Exserohilum, Kocuria,Methylobacterium, Micrococcus, Paenibacillus, Penicillium,Propionibacterium, Pseudomonas, Streptococcus, and Streptomyces.

118. The method of embodiment 117, wherein said Staphylococcus isStaphylococcus aureus, Staphylococcus epidermidis, or Staphylococcushominis.

119. The method of embodiment 117, wherein said Methylobacterium isMethylobacterium radiotolerans.

120. The method of embodiment 117, wherein said Bacillus is Bacillusidriensis, Bacillus licheniformis, or Bacillus substilis.

121. The method of embodiment 117 or 120, wherein said Bacillus isoxidatively stressed.

122. The method of embodiment 117, wherein said Aspergillus isAspergillus brasiliensis or Aspergillus fumigatus.

123. The method of embodiment 117, wherein said Corynebacterium isCorynebacterium tuberculostearicum or Corynebacterium xerosis.

124. The method of embodiment 117, wherein said Dermacoccus isDermacoccus nishinomiyaensis.

125. The method of embodiment 117, wherein said Escherichia isEscherichia coli.

126. The method of embodiment 117, wherein said Kocuria is Kocuriarhizophila.

127. The method of embodiment 117, wherein said Micrococcus isMicrococcus luteus.

128. The method of embodiment 117, wherein said Paenibacillus isPaenibacillus glucanolyticus.

129.The method of embodiment 117: wherein said Penicillium isPenicillium chrysogenum or Penicillium notatum.

130. The method of embodiment 117, wherein said Pseudomonas isPseudomonas aeruginosa.

131. The method of embodiment 117, wherein said Streptococcus isStreptococcus pyogenes.

132. The method of embodiment 117, wherein said Streptomyces isStreptomyces halstedii.

133. The method of embodiment 117, wherein said Acinetobacter isAcinetobacter lwofii.

134. The method of embodiment 117, wherein said Propionibacterium isPropionibacterium acnes.

135. The method of embodiment 117, wherein said Exserohilum isExserohilum rostratum.

136. The method of any one of embodiments 111 to 135, wherein saidpopulation of cells is from a sample.

137. The method of embodiment 136, wherein said sample comprises fluidsor tissues obtained from a multicellular organism.

138.The method of embodiment 137, wherein said sample comprises thebodily fluids or tissues of an animal.

139. The method of embodiment 138, wherein said sample is derived from ahuman.

140. The method of embodiment 138, wherein said sample is derived from anon-human vertebrate.

141. The method of any one of embodiments 137 to 140, wherein saidsample is selected from the group consisting of: respiratory,urogenital, reproductive tract, central nervous system, urine, blood,dermal, plasma, serum, saliva, wound tissue, wound exudate, biopsy,feces, reproductive tract, and solid tissue samples, and derivativesthereof.

142. The method of embodiment 141, wherein said sample is a blood orurine sample.

143. The method of embodiment 137, wherein said sample is derived from aplant

144. The method of any one of embodiments 136 to 143, wherein saidsample is obtained by sampling environmental air, soil, or water, orsurfaces, objects, or organisms exposed to the environment.

145.The method of embodiment 136, wherein said sample is obtained from amaterial selected from the group consisting of raw, finished, orin-process material in the manufacture pharmacological, cosmetic, blood,or other products for topical or internal use in humans or animals; raw,in-process, or finished material in the manufacture of foods, beverages,or nutritional supplements; raw, in-process, or finished material in themanufacture of medical or in vitro diagnostic devices; chemicalproducts; industrial surfaces; instrumentation; and machinery.

146. The method of any one of embodiments 136 to 145, wherein saidsample is treated to liquefy and/or homogenize it prior to saidcontacting.

147. The method of any one of embodiments 136 to 146, wherein saidsample is treated to remove substances or objects other than saidpopulation of cells prior to said contacting.

148 A method of preparing the composition of any one of embodiments 48to 110 comprising:

-   -   i) autoclaving a mixture comprising purified water, casein        digest, soybean digest, a phosphate buffer, dextrose, animal        tissue digest, yeast extract, hemin, and L-cystine;    -   ii) optionally cooling said mixture;    -   iii) optionally adjusting pH to 7.3±0.5 by adding sterile        potassium hydroxide or hydrogen chloride to said mixture; and    -   iv) adding sheep blood to said mixture.

149. The method of embodiment 148, further comprising vi) holding thetemperature of said mixture at about 65° C. until the color of saidmixture changes from red to brown.

150. The method of embodiment 148 or 149, wherein said cooling in stepit) is cooling to room temperature.

151. The method of embodiment 148 or 149, wherein said cooling in stepii) is cooling to about 42° C.

152. The method of any one of embodiments 148 to 151, wherein saidmixture of step i) further comprises a gelling agent.

153. The method of any one of embodiments 148 to 152, wherein saidmixture of step i) further comprises a surfactant.

154. The method of any one of embodiments 148 to 152 further comprisingadding a surfactant to said mixture after step i).

155. The method of embodiment 153 or 154, wherein said surfactant is apolysorbate.

156. The method of any one of embodiment 153 to 155, wherein saidsurfactant is provided as an aqueous solution comprising 5% (w/v) ofsaid surfactant.

157. The method of any one of embodiments 148 to 156 further comprisingtransferring said composition into a storage vessel after preparationsteps are complete.

158. The method of embodiment 157, wherein said storage vessel is abottle, a jar, a vial, an ampoule, or a cassette.

159. The method of embodiment 157 or 158 further comprisingγ-irradiating said storage vessel after said transferring.

160. The method of embodiment 159, wherein the dosage of saidγ-irradiating is greater than 10 kGy.

161. The method of embodiment 160, wherein the dosage of saidγ-irradiating is between about 10 kGy and about 50 kGy.

162. The method of embodiment 161, wherein the dosage of saidy-irradiating is between about 10 kGy and about 40 kGy.

163. The method of embodiment 162, wherein the dosage of saidy-irradiating is between about 10 kGy and about 20 kGy.

164. The method of embodiment 163, wherein the dosage of saidγ-irradiating is between about 12 kGy and about 19 kGy.

165. The composition or method of any of embodiments 1 to 164, whereinthe composition further comprises a disinfectant neutralizer.

166. The composition or method of embodiment 165, wherein theneutralizer is histidine, thiosulfate, polysorbate 80, and/or lecithin.

Other Embodiments

Various modifications and variations of the described composition andmethods of use of the invention will be apparent to those skilled in theart without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the art are intended tobe within the scope of the invention.

Other embodiments are in the claims.

1-47. (canceled)
 48. A method of preparing a composition, comprising:autoclaving a mixture comprising purified water, casein digest, soybeandigest, a phosphate buffer, dextrose, animal tissue digest, yeastextract, hemin, and L-cystine; and adding sheep blood to the mixture.49. The method of claim 48, further comprising cooling the mixture afterautoclaving the mixture.
 50. The method of claim 49, wherein the mixtureis cooled to room temperature.
 51. The method of claim 48, furthercomprising adjusting a pH of the mixture to 7.3±0.5 by adding at leastone of sterile potassium hydroxide or hydrogen chloride to the mixture.52. The method of claim 48, further comprising holding a temperature ofthe mixture at about 65° C. until a color of the mixture changes fromred to brown.
 53. The method of claim 48, wherein the mixture that isautoclaved further comprises a gelling agent.
 54. The method of claim48, wherein the mixture that is autoclaved further comprises asurfactant.
 55. The method of claim 48, wherein the mixture that isautoclaved is free of added sodium.
 56. The method of claim 48, wherein,excluding the phosphate buffer from the total mass of the composition,the composition comprises between about 1 g/kg and about 500 g/kg ofcasein digest.
 57. The method of claim 48, wherein, excluding thephosphate buffer from the total mass of the composition, the compositioncomprises between about 0.5 g/kg and about 300 g/kg of soybean digest.58. The method of claim 48, wherein, excluding the phosphate buffer fromthe total mass of the composition, the composition comprises betweenabout 1 g/kg and about 500 g/kg of animal tissue digest.
 59. The methodof claim 48, wherein, excluding the phosphate buffer from the total massof the composition, the composition comprises between about 1 g/kg andabout 500 g/kg of yeast extract.
 60. The method of claim 48, wherein,excluding the phosphate buffer from the total mass of the composition,the composition comprises between about 1 g/kg and about 500 g/kg ofdextrose.
 61. The method of claim 48, wherein the composition comprisesa quantity of phosphate buffer sufficient to provide a buffer capacityof from 0.1 mmol/(pH unit) to 100 mmol/(pH unit) upon dissolution in anaqueous medium.
 62. The method of claim 48, wherein the mixture that isautoclaved does not comprise tris(hydroxymethyl)aminomethane.
 63. Themethod of claim 48, wherein the mixture that is autoclaved furthercomprises a disinfectant neutralizer.
 64. The method of claim 63,wherein said neutralizer is histidine, thiosulfate, polysorbate 80,and/or lecithin.
 65. The method of claim 48, further comprisingcontacting a population of cells with the composition under conditionssupportive of growth of said population of cells.